A group of excitatory neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b, integrate sensory inputs and information regarding brain state for transmission on to respiratory rhythm/pattern-generating circuits. In addition, RTN neurons adjust their firing in response to changes in CO2 (or H+) and adjust breathing to maintain physiologically appropriate levels of pH and PCO2, a homeostatic process called central respiratory chemoreception. Dysfunction of central chemoreception is implicated in various central disorders of breathing that often occur during sleep (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). In the last project period, we showed that Phox2b-expressing RTN neurons are intrinsically chemosensitive, and identified two independent molecular proton sensors in RTN neurons - TASK-2, a proton-inhibited background K+ channel and GPR4, a proton-activated G protein-coupled receptor - that are required for stimulation of breathing by CO2. Important questions remain, however, regarding: the ionic basis for baseline firing properties and modulation by arousal state-dependent factors; the effector systems engaged downstream of GPR4 in RTN neurons; and mechanisms by proposed astrocytic modulation can be integrated with the requirement for GPR4 and TASK-2 in RTN-mediated respiratory chemosensitivity. This proposal addresses these issues using: novel conditional knockout mouse lines; viral-mediated shRNA knockdown and/or rescue; single cell electrophysiology and molecular biology; and whole animal assays of respiratory function and vigilance states. The hypothesis underpinning Specific Aim 1 is that TTX- resistant subthreshold Na+ channels, NALCN and NaV1.9, contribute to baseline excitability of RTN neurons and mediate facilitatory effects of neuropeptides associated with arousal state-dependent brain nuclei. We disrupt expression of these channels in RTN neurons and determine effects on subthreshold Na+ currents, basal and neuropeptide-modulated firing in vitro, and arousal state-dependent respiratory CO2 sensitivity in vivo. The hypothesis driving Specific Aim 2 is that GPR4 engages a cAMP-transduction pathway and background K+ channel (independent of TASK-2) for cellular pH sensing in RTN neurons, and that astrocytic amplification of respiratory chemoreflexes involves boosting local pH changes around RTN neurons. We use pharmacological and transcriptomic approaches in single RTN neurons to characterize the GPR4 signaling pathway and effector channel, and we disrupt a pH-modulating Na+ -HCO3 transporter, NBCe1, in medullary astrocytes to determine effects on the respiratory chemoreflex in vivo. This latter may support a convergent theory for astrocyte-neuron contributions for this highly sensitive chemoreflex, bridging a major current divide in the field. Collectively, the proposed studies provide critical information regarding molecular and cellular mechanisms that control activity of RTN neurons, and regulate this important homeostatic respiratory system. Identification of novel molecular mechanisms may provide new therapeutic targets for disorders of breathing.